Gene expression modulating element

ABSTRACT

The present invention provides a method of screening for the purpose of obtaining gene expression modulating elements and gene insulator elements. The invention includes a method of identifying gene expression modulating elements and gene insulator elements through use of the following steps: a) locating intergenic regions of a plant genome that are flanked by a gene on each side that have differing gene expressions b) taking that intergenic region or a portion of that intergenic region and adding it to a cassette comprising an isolated gene c) introducing the cassette into a plant cell d) analyzing expression of the isolated gene. The present invention also includes identified sequences that act as gene expression modulating elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/685,347 filed Mar. 13, 2007, now U.S. Pat. No. 7,655,786 issuedFeb. 2, 2010, and claims priority to U.S. Provisional Application Ser.No. 60/782,671 filed Mar. 15, 2006, the disclosure of each isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology andbiotechnology.

BACKGROUND OF THE INVENTION

Transgenic technology is widely used in biotechnology. Systems exist fortransforming plant cells and regenerating complete plants from thetransformed cells; structural gene and gene regulatory regions continueto be identified.

In general, the strength of a given promoter driving a transgene is afunction of its inherent properties and the site of integration.Variation in transgene expression due to the site of integration isoften referred to as “position effect”. It would be useful to have theability to alter the strength of a particular promoter in a predictablemanner.

It is known that eukaryotic genomes have organizational properties thatrely on the ability of the chromosome to establish functional elementsthat are not adversely affected by elements in close proximity.Polynucleotides that decrease the effects of neighboring sequences orgenes are called genetic insulator elements.

SUMMARY OF THE INVENTION

The present invention provides sequences that function as geneexpression modulating elements. It also provides a method to isolatesequences and screen for gene expression modulating elements.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “gene expression modulating element”,“modulating element”, or “modulating sequence” refer to a polynucleotidethat when it is combined with a polynucleotide of interest it does atleast one of the following: a) stabilizes the polynucleotide of interestby decreasing or preventing the influence of other nearby DNA sequencesb) increases the expression of the polynucleotide of interest or c)decreases the expression of the polynucleotide of interest. Whenreferring to “gene expression modulating activity” the activity is thestabilization of, the increasing of, or the decreasing of the expressionof the polynucleotide of interest. When referring to a stabilization ingene expression or an increase or decrease in gene expression, it ismeant when compared to an appropriate control. For example, a control ofa similar sequence size would be used to determine a gene expressionmodulating element. A stabilization in gene expression indicates adecrease in the variability of expression. Variability in expression ofa gene of interest could be influenced by the position of the gene inthe genome and/or by surrounding genes and gene elements such asenhancers, promoters, and terminators.

As used herein, the terms “gene insulator element”, “gene insulator” or“insulator sequence” refer to a polynucleotide that, when it is combinedwith a polynucleotide of interest, stabilizes the polynucleotide ofinterest by decreasing or preventing the influence of other nearby DNAsequences. “Gene insulator activity” is the stabilizing of theexpression of the polynucleotide of interest.

The term “operatively associated,” as used herein, refers to DNAsequences on a single DNA molecule which are associated so that thefunction of one is affected by the other. Thus, a transcriptioninitiation region is operatively associated with a structural gene whenit is capable of affecting the expression of that structural gene (i.e.,the structural gene is under the transcriptional control of thetranscription initiation region). The transcription initiation region issaid to be “upstream” from the structural gene, which is in turn said tobe “downstream” from the transcription initiation region.

“Operably linked” is intended to mean a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame.

“Intergenic region” or “intergenic sequence” is a group of nucleotidesthat lie in tandem and is in between two coding regions. The intergenicregion is not translated.

A “cassette” is a group of nucleotide sequences that lie in tandem. Acassette is usually integrated or exchanged as a unit. For example, aDNA cassette can be the DNA that is used in transformation. It can alsobe the DNA that gets integrated during recombinase-mediated integration.

“Fragments” and “variants” of the nucleotide sequences encodingrecombinases and fragments and variant of recombinase proteins can alsobe used in the present invention. By “fragment” is intended a portion ofthe nucleotide sequence or a portion of the amino acid sequence andhence protein encoded thereby. Fragments of a nucleotide sequence mayencode protein fragments that retain the biological activity of thenative protein and hence implements a recombination event. By “variants”is intended substantially similar sequences. For nucleotide sequences,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode an amino acid sequence thatretains the biological activity of a recombinase polypeptide.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity”, and, (d) “percentage ofsequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

(c) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

As used herein “promoter” is a region of DNA to which an RNA moleculepolymerase and other proteins bind to initiate transcription.

A “marker gene” is a sequence of DNA that when expressed allows it to beidentified. A marker may be a selectable marker gene, a polynucleotideof interest or any gene that produces an identifiable product. Theproduct is either screenable, scorable, visible or detectable. Any genethat produces a protein that can be detected through an ELISA may beconsidered a marker gene. For example, reporter genes, exemplified bychloramphenicol acetyl transferase and beta-glucuronidase (GUS; see,e.g., Jefferson et al. 1987, EMBO J. 6:3901-3907), are commonly used toassess transcriptional and translational competence of chimericconstructions. Other suitable genes include GFP (green florescenceprotein; Chalfie et al. (1994) Science 263:802), luciferase (Riggs etal. (1987) Nucleic Acids Res. 15(19):8115; Luehrsen et al. (1992)Methods Enzymol. 216:397-414) and genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247:449). Standard assays areavailable to sensitively detect marker gene activity in a transgenicorganism.

A “transgene” is a recombinant DNA construct that has been introducedinto the genome by a transformation procedure.

An “isolated” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially or essentially free from componentsthat normally accompany or interact with the nucleic acid molecule orprotein as found in its naturally occurring environment.

An “isolated gene” is free of sequences that naturally flank the gene(i.e., sequences located at the 5′ and 3′ ends of the gene) in thegenomic DNA of the organism from which the gene is derived.

A “gene of interest” is any gene which, when transferred to a plant orplant cell, confers a characteristic. For example any gene that confersvirus resistance, insect resistance, disease resistance, pestresistance, herbicide resistance, improved nutritional value, improvedyield, change in fertility, production of a useful enzyme or metabolitein a plant could be a gene of interest.

A “polynucleotide of interest” is any polynucleotide which, whentransferred to a plant or plant cell, confers a desired characteristic.For example any polynucleotide that confers virus resistance, insectresistance, disease resistance, pest resistance, herbicide resistance,improved nutritional value, improved yield, change in fertility,production of a useful enzyme or metabolite in a plant could be apolynucleotide of interest.

A “selectable marker” is any gene whose expression in a cell gives thecell a selective advantage. The selective advantage possessed by thecells with the selectable marker gene may be due to their ability togrow in the presence of a negative selective agent, such as a antibioticor a herbicide, compared to the ability to grow cells not containing thegene. The selective advantage possessed by the cells containing the genemay also be due to their enhanced capacity to utilize an added compoundsuch as a nutrient, growth factor or energy source.

As used herein a “sexual cross”, “cross” and “sexually crossing”encompass any means by which two haploid gametes are brought togetherresulting in a successful fertilization event and the production of azygote. By “gamete” is intended a specialized haploid cell, either asperm or an egg, serving for sexual reproduction. By “zygote” isintended a diploid cell produced by fusion of a male and female gamete(i.e. a fertilized egg). The resulting “hybrid” zygote containschromosomes from both the acceptor and donor plant. The zygote thenundergoes a series of mitotic divisions to form an embryo.

As defined herein, a “genetically modified plant cell” is a cell thatcomprises a stably integrated DNA sequence of interest.

As defined herein, the “transgenic plant” is a plant that comprises astably integrated DNA sequence of interest.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a modulating sequence or insulator sequence that may be derivedfrom the species of the host cell and is placed in a transgene isconsidered a heterologous sequence due to a location change. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form and/or genomic location.

By “host cell” is meant a cell, which comprises a heterologous nucleicacid sequence of the invention. Host cells may be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells. Optimally, host cells are monocotyledonous ordicotyledonous plant cells. A particularly optimal monocotyledonous hostcell is a maize host cell.

The present invention provides a method of making a transformed plantcell said method comprising: a) providing a plant cell b) transformingsaid plant cell with an isolated nucleic acid wherein said isolatednucleic acid comprises a polynucleotide of interest and a geneexpression modulating element and wherein said gene expressionmodulating element has similar sequence identity to SEQ. ID NO. 1, 2, 3,or 4. An embodiment of the invention is an isolated polynucleotidecomprising SEQ. ID NO. 1, 2, 3, or 4 or a complementary sequencethereof. Another embodiment of this invention is an isolatedpolynucleotide comprising a sequence with 90%, 95%, 97%, 99% identity toSEQ. ID NO. 1, 2, 3, or 4 or a complementary sequence thereof. Anotherembodiment of the invention is an isolated polynucleotide that comprisesthe consensus sequence derived from SEQ. ID NO. 1, 2, 3, or 4. Theisolated polynucleotide comprising the consensus sequence can be 20, 30,or 40 bp to 150 bp but is not limited to these lengths. Anotherembodiment of the present invention is an isolated sequence containingSEQ. ID NO. 1, 2, 3, or 4 that has gene expression modulating element orgene insulator element activity. Another embodiment of the presentinvention is an isolated sequence comprising a combination of portionsof SEQ. ID NO. 1, 2, 3, or 4 that has gene expression modulating elementor gene insulator element activity. Another embodiment is any isolatedpolynucleotide that comprises 20, 30, 40, 50, or 60 continuousnucleotides of SEQ. ID NO. 1, 2, 3, or 4 that has gene expressionmodulating element or gene insulator element activity.

SEQ ID 1: >14-II-2 acaaaattgatctctccatgtagtgttctccacgacgagatctggtgacaactccagtttaagcaagaccaaaagact SEQ ID 2: >5-IV-1ggaccagcgagacagtttatgtgaatgttcatgcttaagtgtcgaacgtatctatctctactatagctctgtagtcttgttagacagttagttttatatctccatttttttgtagtcttgctagttg SEQ ID 3: >5-IV-2ttgctagttgagatattacctcttctcttcaaagtatccttgaacgctcaccggttatgaaatctctacactatagctctgtagtcttgctagatagtta gttctttagctctc SEQ ID4: >5-III-5 attacctcttaaaagtatccttgaacgctttccggttatgaccaatttgttgtagctccttgtaagtagaacttactgggaccagcgagacagtttatgt gaatgt

The present invention provides a method of screening for the purpose ofobtaining gene expression modulating elements and gene insulatorelements. One embodiment is a method of identifying gene expressionmodulating elements and gene insulator elements said method comprising:a) locating intergenic regions of a plant genome that are flanked by agene on each side wherein the first gene has a different expressionpattern than the second gene b) taking said intergenic region or aportion of said intergenic region and adding it to a cassette comprisingan isolated gene c) introducing said cassette into a plant cell d)analyzing expression of said isolated gene in said plant cell. Anotherembodiment of the present invention is said method wherein theintergenic regions are less than 2 kb nucleotides in length. Oneembodiment is said method wherein the intergenic region or a portion ofsaid region is placed in front of the 5′ region of the promoter of theisolated gene, after the promoter and before the coding region of theisolated gene, after the isolated gene, or within the promoter sequence.One embodiment is wherein said intergenic region or a portion of saidintergenic region is placed between the core promoter and the enhancerregion of the isolated gene. One embodiment of the present invention isthe gene expression modulating elements that are derived from saidmethod of screening.

The gene expression modulating element may be located at variouspositions. For example, the modulating element may be located before thepromoter, within the promoter, after the promoter and before the codingregion of the polynucleotide of interest or after the coding region ofthe polynucleotide of interest. The modulating elements can protecttransgenes either from surrounding native influences or can be used toprotect transgenes from interacting with each other in a multigenecassette. The modulating elements can be used to block the influence ofenhancers. The modulating elements can be placed between two genesadjacent to each other for setting clear boundary between two promotersand prevent unwanted interference of gene expression. The non-codingintergenic DNA elements are useful for improving and solving currentobstacles such as transcriptional interference, chromatin repression,expression variegation for high transformation efficiency and transgenicplants recovery.

Introducing in the present invention can be through transienttransformation, stable transformation, and/or through DNA integrationrecombinase systems.

Intergenic regions of a plant genome wherein the first gene has adifferent expression pattern than the second gene can be located usingany combination of bioinformatics tools, molecular maps, and expressiondata. Expression data can be obtained through various means such asWestern analysis, microarray analysis, Northern blots, nuclear run onanalysis, RT-PCR, quantitative real-time RT-PCR, and public databases(Pennisi, Science 286, 449 (1999); Kaiser, (ed.) Science 286, 1047(1999)). Different expression patterns can be different at differenttimes, throughout the development of the plant, in response toenvironment changes, and/or due to cell type. Different expressionpatterns may be consistent over time and development but may bedifferent in amounts of expression only. The amount of product expressedby the two genes flanking the intergenic region can vary. For the twogenes to be considered as having different expression patterns theamount of product difference, RNA or protein, may be 100 ppm or greater,100 ppm to 1000 ppm, or 1000 ppm or greater at any point in time.

The length of the intergenic region in this invention can be less than,3 kb, less than 2 kb, less than 1.75 kb, less than 1.5 kb or less than 1kb. The length of the intergenic region can be from 20 base pairs to 2kb, 30 base pairs to 2 kb, 20 base pairs to 1.75 kb, 30 base pairs to1.75 kb, 20 base pairs to 3 kb, 30 base pairs to 3 kb, 40 base pairs to1.5 kb, and 40 base pairs to 2 kb.

The intergenic region can be divided into portions for screening. Theseportions can be any length from 20 bp to about 1 kb in length.

The present invention includes gene expression modulating elements andgene insulator elements. One embodiment is gene expression modulatingelements and gene insulator elements that range in length from 20 to 200bp, from 20 to 150 bp, from 20 to 100 bp, from 30 to 200 bp, and from 40to 200 bp.

The methods of the invention can be carried out with cells from avariety of different plant cells. The cells may be monocots or dicots.Examples of plant species of interest include, but are not limited to,corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Embodiments of this invention include sequences that have 80%, 85%, 90%,95%, 97%, 98%, 99% and 100% identity to SEQ. ID NO. 1, 2, 3, or 4 andisolated nucleic acids fully complementary thereof. Methods of alignmentof sequences for comparison are well known in the art. Thus, thedetermination of percent sequence identity between any two sequences canbe accomplished using a mathematical algorithm. Non-limiting examples ofsuch mathematical algorithms are the algorithm of Myers and Miller(1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al.(1981) Adv. Appl. Math. 2:482; the global alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Embodiments ofthis invention include fragments and combinations of fragments among theSEQ ID NO. 1, 2, 3, and 4.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the defaultparameters of the respective programs (e.g., BLASTN for nucleotidesequences, BLASTX for proteins) can be used. See the world wide webncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3; or anyequivalent program thereof. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. For nucleotide sequences the default gap creationpenalty is 50 while the default gap extension penalty is 3. The gapcreation and gap extension penalties can be expressed as an integerselected from the group of integers consisting of from 0 to 200. Thus,for example, the gap creation and gap extension penalties can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

There are many genes of interest or polynucleotides of interest that canbe used in transgenes and therefore can be used in this invention.Polynucleotides of interest that when expressed can confer herbicideresistance, insect resistance, disease resistance, drought tolerance,male sterility, restoration of male fertility, amino acid changes,nutritional improvements, agronomic improvements, changes in maturity,increase in yield, changes in flowering time, increases in protein,increases in starch, decreases in starch, changes in phytate content,increase in oil, change in the oil content, or increases intransformation efficiency. Exemplary polynucleotides of interestimplicated in this regard include, but are not limited to, thosecategorized below.

1. Transgenes that Confer Resistance to Pests or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae). A plant resistant to a disease is one that ismore resistant to a pathogen as compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998. Other examples of Bacillus thuringiensis transgenes beinggenetically engineered are given in the following patents and hereby areincorporated by reference for this purpose: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; and WO 97/40162.

(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(D) A vitamin-binding protein such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by referencefor this purpose. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987)(nucleotide sequence of rice cysteine proteinase inhibitor), Huub etal., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor) and U.S. Pat. No. 5,494,813.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTApplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT Application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT Application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference for this purpose.

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89:43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-induced resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus.

(K) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

(L) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(M) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology5:128-131 (1995).

(N) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998).

2. Transgenes that Confer Resistance to an Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate which has resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively. See, for example, U.S. Pat. No. 4,940,835 to Shah et al.,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry et al.also describes genes encoding EPSPS enzymes. See also U.S. Pat. Nos.6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908;5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE37,287 E; and 5,491,288; and international publications WO 97/04103; WO97/04114; WO 00/66746; WO 01/66704; WO 00/66747 and WO 00/66748, whichare incorporated herein by reference for this purpose. Glyphosateresistance is also imparted to plants that express a gene that encodes aglyphosate oxido-reductase enzyme as described more fully in U.S. Pat.Nos. 5,776,760 and 5,463,175, which are incorporated herein by referencefor this purpose. In addition glyphosate resistance can be imparted toplants by the over expression of genes encoding glyphosateN-acetyltransferase (GAT). See, for example, PCT publication WO02/36782and U.S. application Ser. No. 10/427,692. A DNA molecule encoding amutant aroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai.

(C) Phosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) genes. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al., De Greef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. European Patent Application No. 0 333 033 toKumada et al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides such as L-phosphinothricin. See also, U.S. Pat.Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 B1; and 5,879,903, which areincorporated herein by reference for this purpose.

(D) Pyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

(E) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

(F) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet. 246:419). Other genes that confer tolerance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, and genes forvarious phosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619).

(G) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and International Publication WO 01/12825, which areincorporated herein by reference for this purpose.

3. Transgenes that Confer or Contribute to a Grain Trait, Such as:

(A) Modified fatty acid metabolism, for example, by transforming a plantwith a gene that suppresses stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2624 (1992).

(B) Phytate content

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127:87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) A gene could be introduced that reduces phytate content.        Examples of genes are disclosed in U.S. Pat. Nos. 6,197,561;        6,291,224 and WO 02/059324.

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10:292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II). U.S. Pat.Nos. 6,43,886 and 6,399,859 disclose starch synthase genes in maize.

(D) Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification (see U.S. Pat. Nos.6,063,947; 6,323,392; and WO 93/11245).

4. Genes that Control Male-Sterility:

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al., PlantMol. Biol. 19:611-622, 1992).

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acid can be combined with constitutive, tissue-preferred,developmentally regulated, or other promoters for expression in plants.Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611. The Cauliflower MosaicVirus 35S promoter is one of the promoters used most often for dicottransformation because it confers high levels of gene expression inalmost all tissues (J. Odell et al., 1985; D. W. Ow et al., 1986; D. M.Shah et al., 1986). Modifications of this promoter are also used,including a configuration with two tandem 35S promoters (R. Kay et al.,1987) and the mas-35S promoter (L. Comai et al., 1990), which consistsof the mannopine synthase promoter in tandem with the 35S promoter. Bothof these promoters drive even higher levels of gene expression than asingle copy of the 35S promoter. Other viral promoters that have beenused include the Cauliflower Mosaic Virus 19S promoter (J. Paszkowski etal., 1984; E. Balazs et al.) and the 34S promoter from the figwortmosaic virus (M. Sanger et al., 1990).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced ODP2expression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen.Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and, milps(myo-inositol-1-phosphate synthase); (see WO 00/11177 and U.S. Pat. No.6,225,529; herein incorporated by reference). Gamma-zein is anotherendosperm-specific promoter (Boronat et al. (1986) Plant Science47:95-102). Globulin-1 (Glob-1) is a preferred embryo-specific promoter.For dicots, seed-specific promoters include, but are not limited to,bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, andthe like. For monocots, seed-specific promoters include, but are notlimited to, maize 15 kDa, 22 kDa zein, 27 kDa zein, gamma-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. Additional seed-preferred promoters includethe oleosin promoter (WO 00/0028058), the lipid transfer protein (LTP)promoter (U.S. Pat. No. 5,525,716). Additional seed-preferred promotersinclude the Led promoter, the Jip1 promoter, and the milps3 promoter(see, WO 02/42424). Led-indicates a leafy cotyledon 1 transcriptionalactivator polynucleotide. See U.S. patent application Ser. No.09/435,054. Lec1 promoter is characterized in U.S. Pat. No. 7,122,658.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the polynucleotide of interest andtherefore influenced by modulating elements and insulating elements.

The methods of the invention involve introducing a nucleotide constructor a polypeptide into a plant. By “introducing” is intended presentingto the plant the nucleotide construct (i.e., DNA or RNA) or apolypeptide in such a manner that the nucleic acid or the polypeptidegains access to the interior of a cell of the plant. The methods of theinvention do not depend on a particular method for introducing thenucleotide construct or the polypeptide to a plant, only that thenucleotide construct gains access to the interior of at least one cellof the plant. Methods for introducing nucleotide constructs and/orpolypeptides into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods,virus-mediated methods, DNA integration recombinase systems.

By “stable transformation” is intended that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide construct or thepolypeptide introduced into a plant does not integrate into the genomeof the plant.

In preparing a DNA cassette, various DNA fragments may be manipulated,so as to provide for the DNA sequences in the proper orientation and, asappropriate, in the proper reading frame. Toward this end, adapters orlinkers may be employed to join the DNA fragments or other manipulationsmay be involved to provide for convenient restriction sites, removal ofsuperfluous DNA, removal of restriction sites, or the like. For thispurpose, in vitro mutagenesis, primer repair, restriction, annealing,resubstitutions, e.g., transitions and transversions, may be involved.The DNA cassettes may additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example, EMCVleader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al.(1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods or sequences known to enhancetranslation can also be utilized, for example, introns, and the like.

The method of transformation is not critical to the invention; variousmethods of transformation are currently available. As newer methods areavailable to transform host cells they may be directly applied.Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence. Thus, any method that provides forefficient transformation/transfection may be employed.

Methods for transforming various host cells are disclosed in Klein etal. “Transformation of microbes, plants and animals by particlebombardment”, Bio/Technol. New York, N.Y., Nature Publishing Company,March 1992, 10(3):286-291. Techniques for transforming a wide variety ofhigher plant species are well known and described in the technical,scientific, and patent literature. See, for example, Weising et al.,Ann. Rev. Genet. 22:421-477 (1988).

For example, the DNA construct may be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporation,PEG-induced transfection, particle bombardment, silicon fiber delivery,or microinjection of plant cell protoplasts or embryogenic callus. See,e.g., Tomes et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods. eds. O. L. Gamborg and G. C. Phillips.Springer-Verlag Berlin Heidelberg New York, 1995. The introduction ofDNA constructs using polyethylene glycol precipitation is described inPaszkowski et al., Embo J. 3:2717-2722 (1984). Electroporationtechniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82:5824(1985). Ballistic transformation techniques are described in Klein etal., Nature 327:70-73 (1987).

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a Agrobacterium tumefaciens hostvector. The virulence functions of the Agrobacterium tumefaciens hostwill direct the insertion of the construct and adjacent marker into theplant cell DNA when the cell is infected by the bacteria. Agrobacteriumtumefaciens-meditated transformation techniques are well described inthe scientific literature. See, for example Horsch et al., Science233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803(1983). For instance, Agrobacterium transformation of maize is describedin U.S. Pat. No. 5,981,840. Agrobacterium transformation of monocot isfound in U.S. Pat. No. 5,591,616. Agrobacterium transformation ofsoybeans is described in U.S. Pat. No. 5,563,055.

Other methods of transformation include (1) Agrobacteriumrhizogenes-induced transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press,1987; and Lichtenstein, C. P., and Draper, J., In: DNA Cloning, Vol. II,D. M. Glover, Ed., Oxford, IRI Press, 1985), Application PCT/US87/02512(WO 88/02405 published Apr. 7, 1988) describes the use of A. rhizogenesstrain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 orpARC16 (2) liposome-induced DNA uptake (see, e.g., Freeman et al., PlantCell Physiol. 25:1353, 1984), (3) the vortexing method (see, e.g.,Kindle, Proc. Natl. Acad. Sci., USA 87:1228, (1990).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol. 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingnucleic acids can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena et al., Nature 325:274 (1987).Transformation can also be achieved through electroporation of foreignDNA into sperm cells then microinjecting the transformed sperm cellsinto isolated embryo sacs as described in U.S. Pat. No. 6,300,543 byCass et al. DNA can also be injected directly into the cells of immatureembryos and the rehydration of desiccated embryos as described byNeuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook et al.,in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54(1986).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell 2:603-618 (1990).

Introducing a polynucleotide sequence into a cell can be achieved usinga DNA integration recombinase system. DNA integration recombinasesystems involve DNA cassettes one, which can be identified as “donorDNA” and one, which can be identified as “target DNA”. The target DNAgenerally comprises at least two recombinase recognition sites. Thesites flank a polynucleotide that may comprise a gene or a set of geneexpression cassettes. In the present invention the recombinationrecognition sites can be identical and/or non-identical. The donor DNAgenerally comprises at least two recombinase recognition sites. Thesites flank a polynucleotide that may comprise a gene or a set of geneexpression cassettes. DNA integration recombinase systems also have oneor more proteins, called recombinases, which mediate the specificcleavage and ligation of the recombinase recognition sites. Therecombinases can enter the system in various ways. For instance, apolynucleotide encoding the recombinase could be within the target DNA,the donor DNA, within the genome of a target plant, or within the genomeof the donor plant. The recombinase could also enter the system viatransient expression or as an active recombinase. The donor DNA can beinitially integrated into the plant cell through transformation. Afterthe donor DNA has been stably integrated into the plant cell, moregenetically modified cells can be propagated from the transformed plantcell or plants can be obtained from the transformed plant cells and thedonor DNA can be inherited via sexual and asexual reproduction. Thetarget DNA can also be initially integrated into the plant cell throughtransformation. After the target DNA is stably integrated into the plantcell more genetically modified cells can be propagated from thetransformed plant cell or plants can be obtained from the transformedplant then cells and the target DNA can be inherited via sexual andasexual reproduction.

After the donor DNA and the target DNA have been stably integrated intoseparate plants, creating a donor plant and a target plant, the plantsthen can be sexually crossed. Recombinase-mediated integration can occurwith the crossing of the donor plant and the target plant in thepresence of corresponding recombinase. The term “crossing” does notdesignate which plant is to be used as a male and which plant is to beused as a female, thus for purposes of this invention the plantcontaining the target DNA can be used as either the male or female inthe cross.

The donor DNA and the target DNA can also be brought together throughtransformation of cells. If the donor DNA is stably integrated into acell, the target DNA can then be used to transform the cell. In thepresence of corresponding recombinase, recombinase-mediated integrationcan occur. If the target DNA is stably integrated into a cell, the donorDNA then can be used to transform the cell. Once again in the presenceof corresponding recombinase, recombinase-mediated integration canoccur.

Examples of recombination sites for use in the invention are known inthe art and include FRT sites (See, for example, U.S. Pat. No.6,187,994; Schlake and Bode (1994) Biochemistry 33:12746-12751; Huang etal. (1991) Nucleic Acids Research 19:443-448; Paul D. Sadowski (1995) InProgress in Nucleic Acid Research and Molecular Biology 51:53-91;Michael M. Cox (1989) In Mobile DNA, Berg and Howe (eds) AmericanSociety of Microbiology, Washington D.C., pp. 116-670; Dixon et al.(1995) 18:449-458; Umlauf and Cox (1988) The EMBO Journal 7:1845-1852;Buchholz et al. (1996) Nucleic Acids Research 24:3118-3119; Kilby et al.(1993) Trends Genet. 9:413-421; Rossant and Geagy (1995) Nat. Med.1:592-594; Albert et al. (1995) The Plant J. 7:649-659; Bayley et al.(1992) Plant Mol. Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet.223:369-378; and Dale and Ow (1991) Proc. Natl. Acad. Sci. USA88:10558-105620; all of which are herein incorporated by reference); lox(Albert et al. (1995) Plant J. 7:649-659; Qui et al. (1994) Proc. Natl.Acad. Sci. USA 91:1706-1710; Stuurman et al. (1996) Plant Mol. Biol.32:901-913; Odell et al. (1990) Mol. Gen. Gevet. 223:369-378; Dale etal. (1990) Gene 91:79-85; and Bayley et al. (1992) Plant Mol. Biol.18:353-361.) Dissimilar recombination sites are designed such thatintegrative recombination events are favored over the excision reaction.Such dissimilar recombination sites are known in the art. For example,Albert et al. introduced nucleotide changes into the left 13 bp element(LE mutant lox site) or the right 13 bp element (RE mutant lox site) ofthe lox site. Recombination between the LE mutant lox site and the REmutant lox site produces the wild-type loxP site and a LE+RE mutant sitethat is poorly recognized by the recombinase Cre, resulting in a stableintegration event (Albert et al. (1995) Plant J. 7:649-659). See also,for example, Araki et al. (1997) Nucleic Acid Research 25:868-872.

Various recombinases can be used in this invention. For reviews ofsite-specific recombinases, see Sauer (1994) Current Opinion inBiotechnology 5:521-527; and Sadowski (1993) FASEB 7:760-767; thecontents of which are incorporated herein by reference. The recombinaseused in the methods of the invention can be a naturally occurringrecombinase or an active fragment or variant of the recombinase.Recombinases useful in the methods and compositions of the inventioninclude recombinases from the Integrase and Resolvase families,biologically active variants and fragments thereof, and any othernaturally occurring or recombinantly produced enzyme or variant thereof,that catalyzes conservative site-specific recombination betweenspecified DNA recombination sites. The Integrase family of recombinaseshas over one hundred members and includes, for example, FLP, Cre, Intand R. For other members of the Integrase family, see for example,Esposito et al. (1997) Nucleic Acid Research 25:3605-3614 and Abremskiet al. (1992) Protein Engineering 5:87-91, both of which are hereinincorporated by reference. Such recombination systems include, forexample, the streptomycete bacteriophage phi C31 (Kuhstoss et al. (1991)J. Mol. Biol. 20:897-908); the SSV1 site-specific recombination systemfrom Sulfolobus shibatae (Maskhelishvili et al. (1993) Mol. Gen. Genet.237:334-342); and a retroviral integrase-based integration system(Tanaka et al. (1998) Gene 17:67-76). In other embodiments, therecombinase is one that does not require cofactors or a supercoiledsubstrate. Such recombinases include Cre, FLP, or active variants orfragments thereof. See U.S. Pat. No. 5,929,301.

The FLP recombinase is a protein that catalyzes a site-specific reactionthat is involved in amplifying the copy number of the two-micron plasmidof S. cerevisiae during DNA replication. The FLP recombinase catalyzessite-specific recombination between two FRT sites. The FLP protein hasbeen cloned and expressed. See, for example, Cox (1993) Proc. Natl.Acad. Sci. USA 80:4223-4227. The FLP recombinase for use in theinvention may be that derived from the genus Saccharomyces. One can alsosynthesize the recombinase using plant-preferred codons for optimalexpression in a plant of interest. A recombinant FLP enzyme encoding bya nucleotide sequence comprising maize preferred codons (moFLP) thatcatalyzes site-specific recombination events is known. See, for example,U.S. Pat. No. 5,929,301, herein incorporated by reference. Additionalfunctional variants and fragments of FLP are known. See, for example,Hartung et al. (1998) J. Biol. Chem. 273:22884-22891 and Saxena et al.(1997) Biochim Biophys Acta 1340(2):187-204, and Hartley et al. (1980)Nature 286:860-864, all of which are herein incorporated by reference.

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites. The Cre recombinase is known in the art. See, forexample, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J.Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet.22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702, allof which are herein incorporated by reference. The Cre sequences mayalso be synthesized using plant-preferred codons. Such sequences (moCre)are described in WO 99/25840, herein incorporated by reference.

It is further recognized that chimeric recombinases can be used in themethods of the present invention. By “chimeric recombinase” is intendeda recombinant fusion protein which is capable of catalyzingsite-specific recombination between recombination sites that originatefrom different recombination systems. That is, if the non-identicalrecombination sites utilized in the present invention comprise FRT andloxP sites, a chimeric FLP/Cre recombinase or active variant thereofwill be needed or both recombinases may be separately provided. Methodsfor the production and use of such chimeric recombinases or activevariant thereof are described in U.S. Pat. No. 6,262,341 and U.S. Pat.No. 6,541,231, herein incorporated by reference.

EXAMPLES Example 1

Available MPSS (Massively Parallel Signature Sequencing) data for theArabidopsis genome was searched for intergenic regions that couldpotentially contain modulator elements. This data is available at apublic website (mpss.udel.edu). MPSS is a technique described by Solexa,Inc. of Hayward, Calif. MPSS and related technologies have beendescribed in publications by Brenner et al. (Nature Biotechnol. 2000,18: 630-634, and PNAS, 2000, 97:1665-1670).

The following criteria were used to survey the Arabidopsis genome forpotential modulating element sequences. First, tandemly arranged genesthat have relatively short intergenic regions were located. Theexpression pattern of the individual genes in the tandem pairs wasdetermined using Arabidopsis genome annotation information (TIGR ver 3)and parsing program (Phyton ver 2.3) with some modifications forexperimental purposes. The intergenic regions flanked by tandem pairs ofgenes with different expression patterns were considered further. Basalor core promoter regions that have a TATA Box and previously knowncis-elements were disregarded. As a result of this bioinformaticanalysis, a locus containing two hsc70 (heat shock) genes separated by1.4 Kb of intergenic space in Arabidopsis chromosome 5 was found. Thisintergenic region was chosen for further analysis.

For analysis of modulating elements, particle bombardment was used todeliver test constructs into embryogenic tissue cultures of soybean. Thetest constructs were designed to determine the ability of putativemodulating elements to block enhancer action on a core promoter.Therefore, the candidate element was cloned between 35S enhancers andcore promoter. This 5′ region was used to control expression of afirefly luciferase gene. A Renilla lucierase gene under control of themaize ubiquitin promoter served as an internal control. About 24 hoursafter bombardment, luciferase expression was determined for theconstruct with the candidate modulator and for the co-introducedubiquitin construct.

Two candidate DNA regions, a 1.4 kb intergenic region (hsc14) andapproximately 3 kb of a 5′ upstream region (hsc5) of the hsc70 gene weresubcloned as approximately 500 bp fragments into the assay vector. Theelements were cloned into the assay vector in both forward and reverseorientation. The vectors were introduced into soybean embryogenicsuspensions by particle bombardment and luciferase activity wasdetermined after about 24 hours. Table 1 shows luciferase activityderived from the assay vectors. Some of the elements such as 5-II, 5-IIIand 14-IV substantially blocked expression from the 35S enhancers.

Three DNA fragments (hsc14-II, hsc5-III and hsc5-IV) were selected forfurther analysis. These fragments were dissected into approximately 100bp sequences and cloned into the assay vector. Three of theapproximately 100 bp fragments (5-II-5,5-IV-1, and 5-IV-2) showed strongenhancer blocking activity (Table 2). Luciferase expression with the5-III-5,5-IV-1, and 5-IV-2 containing constructs was 20% to 30% of theexpression exhibited in the control. Controls were the 35S enhancer andcore promoter without any candidate sequences between them.

The impact of four of the putative modulator sequences (5-III-5, 5-IV-1,5-IV-2, and 14-II-2) were then assessed in transgenic cell linesgenerated by stable transformation of embryogenic tissue cultures. Toaccomplish this, the assay construct was introduced with a selectablemarker gene that confers resistance to hygromycin. The construct usedfor these experiments is shown below. Transgenic events were recoveredeight weeks after bombardment and the luciferase activity in each eventwas analyzed (Table 3). The effect of the modulator on 35S enhanceractivity was compared to luciferase activity from events harboring a35S/luciferase control construct. Fragments 5-III-5 and 5-IV-2 appearedto substantially block expression from the 35S enhancers.

Construct used for analysis of modulator elements in stably transformedsoybean tissue.

Nos 3′--LUC-F--35S Core-Candidate seq.-35S En----Nos 3′-Hyg-35SPro                                    ←                          ←

TABLE 1 First round screening of modulating and insulator elements(Percentages are based on lucifease activity measured in LUX units)Orientation Forward Orientation Reverse Orientation SEQ ID % of ControlSD % of Control SD  5-I 16.47 0.96 64.23 18.18  5-II 5.77 2.43 34.297.10  5-III 9.28 1.94 14.36 0.21  5-IV 13.98 3.45 18.14 2.98  5-V 42.3311.97 71.13 3.46  5-VI 49.36 53.97 54.35 20.64 14-I 36.18 2.11 57.5232.01 14-II 23.39 7.09 61.23 28.41 14-III 36.35 9.58 44.17 21.79 14-IV9.83 3.55 28.36 14.19

TABLE 2 Second round screening of modulating and insulator elementsOrientation Forward Orientation Reverse Orientation SEQ ID % of ControlSD % of Control SD  5-III-1 33.74 5.36 35.78 6.35  5-III-3 50.06 7.9367.10 19.80  5-III-4 31.03 9.26 41.87 4.68  5-III-5 26.53 5.05 33.925.76  5-IV-1 39.34 14.53 24.37 6.29  5-IV-2 23.01 5.54 43.80 11.66 5-IV-3 41.37 9.32 32.78 4.44  5-IV-4 61.09 17.19 47.90 22.02  5-IV-587.80 6.74 83.02 19.60 14-II-1 99.63 35.84 76.43 29.53 14-II-2 63.3618.93 84.69 41.55 14-II-3 71.73 19.70 59.10 31.68 14-II-4 138.43 46.73147.02 61.76 14-II-5 97.49 25.77 157.55 47.52

TABLE 3 Gene expression modulating and insulator activity of selectedDNA fragments in stable transformation measured in LUX Control 5-III-55-IV-1 5-IV-2 14-II-2 Forward Orientation Average 12223.4 2081.753713.02% 2799.51% 3645.24% 17.03% 30.38% 22.90% 29.82% Median 4344.59746.462 565.954 552.149 1015.64 17.18% 13.03% 12.71% 23.38% ReverseOrientation Average 9718.42 4625.82 5819.41 3706.221 2977.134 47.60%59.88% 38.14% 30.63% Median 4546.53 1715.64 1361.15 1621.15 1400.5937.74% 29.94% 35.66% 30.81%

Example 2

The modulators identified in the present work block the ability ofenhancer elements to activate a core promoter. It is know in theliterature that enhancers for constitutive activation of one promoter ina transgene cassette can inadvertently activate other promoters thatshould provide tissue specific expression. For example, the 35Senhancers controlling the expression of a marker gene can activate theexpression of a co-delivered tissue specific gene. See Yoo et al. (2005)Planta 221:523-530. Table 4 provides an example of this phenomenon inmaize. Construct 1 in Table 4 contains a bar gene under the control ofthe 35S promoter and associated enhancers. This construct also containsa gus gene under the control of the oleosin promoter. Oleosin normallyprovides expression in certain seed tissue such as the scutellum. Thisconstruct was introduced into maize by agrobacterium-mediatedtransformation. Plants were recovered and gus expression was analyzed inleaf tissue. These plants had an average GUS expression of 284 pMole MUu. This demonstrates that the 35S enhancers in the construct producedun-wanted expression of the oleosin promoter in leaves. Similarobservations were made with constructs 2 and 3 in Table 2. Unlike the35S promoter, the ubiqutin promoter in construct 4 did not activate theoleosin promoter in leaves. Two constructs harboring a modulatingelement (AT-5-IV-2) between the oleosin/GUS gene and 35S/bar gene wereintroduced into maize (constructs 5 and 6). Transgenic events with theseconstructs had low levels of GUS expression, far lower than controlslacking the modulator element. These results show that the modulator canblock unwanted activation of tissue specific promoters.

pinII -indicates potato proteinase inhibitor. See Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498.

Ubi indicates a ubiquitin promoter. See Christensen et al. (1989) PlantMol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689).

BAR indicates Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) genes. Leemans et al., De Greef et al., Bio/Technology7:61 (1989), describe the production of transgenic plants that expresschimeric bar genes coding for phosphinothricin acetyl transferaseactivity; genes which confer resistance to herbicides such asL-phosphinothricin.

MoPAT indicates a modified pat gene (phosphinothricin acetyl transferase(PAT)) which gives resistance glufosinate).

ADH1 indicates an alcohol dehydrogenase (Adh) gene promoter (see, e.g.,Millar (1996) Plant Mol. Biol. 31:897-904).

OLE indicates a oleosin promoter (WO 00/0028058).

NOS indicates the nopaline synthase (nos) promoter (M. W. Bevan et al.,1983; L. Herrera-Estrella et al., 1983, R T. Fraley et al., 1983, M. DeBlock et al., 1984; R Hain et al., 1985).

35S indicates the Cauliflower Mosaic Virus 35S promoter (J. Odell etal., 1985; D. W. Ow et al., 1986; D. M. Shah et al., 1986).

GUS indicates a beta-glucuronidase gene (see, e.g., Jefferson et al.1987, EMBO J. 6:3901-3907)

Corn plants were transformed using Agrobacterium transformation. Sixdifferent expression cassettes were used (Table 4).

TABLE 4 AVG GUS Score in Leaf Range of GUS CASSETTE (pMole_MU_u)Score 1) NOS:GUSINT:OLE 0.9 KB PRO//35S:ADH1 INTRON:BAR:PINII 284 (13plants) 0-795 2) NOS:GUSINT:OLE//35S:ADH1 INTRON:BAR:PINII 1165 (2003data) N/A 3) OLE 0.9 KB PRO:GUSINT:NOS//35S:ADH1 INTRON:BAR:PINII 237(2003 data) N/A 4) OLE 0.9 KB PRO:GUSINT:NOS//UBI:UBI INTRON:MOPAT:35S 0(2003 data) N/A TERM 5) OLE:GUSINT:NOS//AT-5-IV-2//35S:O′:ADH1INTRON:BAR:PINII 31 (11 plants) 18-40  6) NOS:GUSINT:OLE PRO//AT-5-IV-2//35S:O′:ADH1 79 (15 plants) 0-220 INTRON:BAR:PINII

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid comprising a gene expression modulatingelement operably linked to a heterologous promoter and a polynucleotideof interest, wherein said gene expression modulating element is SEQ. IDNO. 2, and said gene modulating element influences the expression of thepolynucleotide of interest.
 2. A transformed plant cell comprising thenucleic acid of claim
 1. 3. The plant cell of claim 2 wherein said plantcell is a corn, soybean, sorghum, sunflower, safflower, wheat, rice,alfalfa, or Brassica cell.
 4. A transgenic plant comprising the nucleicacid of claim
 1. 5. The transgenic plant of claim 4 wherein said plantis corn, soybean, sorghum, sunflower, safflower, wheat, rice, alfalfa,or Brassica.
 6. A transgenic seed from the plant of claim 4, wherein thetransgenic seed comprises the nucleic acid.
 7. The transgenic seed ofclaim 6 wherein said seed is from corn, soybean, sorghum, sunflower,safflower, wheat, rice, alfalfa, or Brassica.
 8. The isolated nucleicacid of claim 1 wherein said promoter comprises a 35S promoter.
 9. Theisolated nucleic acid of claim 1 wherein said promoter comprises aubqiuitin promoter.
 10. The isolated nucleic acid of claim 1, whereinthe polynucleotide of interest comprises a coding region.
 11. Theisolated nucleic acid of claim 10, wherein the gene modulating elementof SEQ ID NO: 2 is located before the promoter.
 12. The isolated nucleicacid of claim 10, wherein the gene modulating element of SEQ ID NO: 2 islocated after the coding region.
 13. The isolated nucleic acid of claim10, wherein the gene modulating element of SEQ ID NO: 2 is locatedbetween the promoter and the coding region.
 14. The isolated nucleicacid of claim 1, wherein the polynucleotide of interest comprises a genethat confers insect resistance.